Most cellular processes, such as DNA replication, transcription and chromosome dynamics, are regulated by binding of protein complexes to specific cis-acting elements of chromatin templates.
Many of these interactions have recently been studied with the aid of the chromatin immunoprecipitation technique (ChIP), which is a versatile, widely used method to localize bound proteins to genomic loci with a resolution of about 1 kb (reviewed by Orlando, 2000). ChIP is based on formaldehyde fixation of cells followed by immunoprecipitation of fragmented chromatin with specific antibodies. One basic assumption of ChIP is that immunoprecipitation is applied to soluble (or solubilized) chromatin that consists of linear or specifically branched, not randomly crosslinked, chromatin fragments. Toward this goal, fixed cells are exposed to a combined action of sonication (sometimes nucleases) and denaturing agents. This fragmentation-solubilization step is followed by centrifugation to remove large insoluble material prior to the immunoprecipitation step. Although rarely stated, many structural-type chromatin proteins remain quite insoluble and significant amounts are lost into the pellet during centrifugation.
The ChIP method is afflicted with several problems and limitations. Often this method yields results associated with a high background, where the signal to noise ratio is low. This background has different causes such as, limited antibody specificity, non-specific chromatin aggregation and fixation artifacts. The chemical fixation step, usually by formaldehyde, is necessary as to ‘freeze’ proteins of interest to chromatin to allow fragmentation by sonication. But fixation is an uncontrolled step, that also leads to non-specific crosslinking of chromatin fragments into branched aggregates, it also yields ‘nuclear crumbs’ by shattering the fixed nucleus by sonication that are not removed by the centrifugation step. Hence, the basic assumption, unbranched chromatin fragments, is not necessarily met.
In consequence, the data obtained by ChIP, an in vitro method, do not necessary reflect the in vivo situation.
Moreover, ChIP analyses with insoluble-type proteins appear afflicted with increased background, supposedly arising by non-specific crosslinking of chromatin into branched aggregates or ‘nuclear crumbs’.
An alternative technique for identification of DNA loci that interact in vivo with specific nuclear proteins is named DamID (van Steensel and Henikoff, 2000). In this method, the adenine methyltransferase (Dam) for E. coli is expressed as a fusion protein, X-Dam, in vivo. X is a protein of interest bound to chromatin that one wants to genomically map. The targeted, chromatin bound X-Dam, methylates in vivo adenine (N6 position) at GATC sequences, which occur every 200-300 bp. A restriction enzyme, DpnII, is then used to measure the extent of methylation. This enzyme cleaves the DNA, if a Dam site is unmethylated, while it does not cleave, if the site is methylated. Hence, to map a X-Dam protein genomically, one measures around putative genomic targets of X, the relative cleavability of Dam sites using either PCR and Southern blotting.
Main disadvantages of DamID are poor resolution and unregulated expression. First, Dam sites only occur on average every 200-300 bp, so sampling distances are large; the DNA in-between remains unsampled. Second, since methylation occurs continuously, it is not regulated ON/OFF, methylation occurs in a large DNA neighbourhood (several kb) surrounding genomic binding sites for X.
Another alternative method, which only works in vivo, is based on the technique named “PIN*POINT” (Lee et al, 1998). According to this technique, a fusion gene product X-FokI is expressed in the cell, wherein X is the chromatin protein under study and FokI stands for the nuclease moiety of the endonuclease FokI. The binding of the X protein to DNA is made detectable by the nuclease-induced cleavage near the binding site of X, by primer extension or ligation-mediated PCR.
However, expression of an active, non-regulatable nuclease in vivo is very damming to the cell. In vivo DNA breaks accumulated during the entire expression period. This induces the DNA damage checkpoint pathway, which alters the cell physiology dramatically.
Therefore, in view of the drawbacks of the available techniques there is a need for a technique which combines a high resolution mapping (at most around 100 base pairs or even less) with confidence and also a high sensitivity to biological changes without interferences by artefacts linked to the method.
The technique must also preferably be adapted for in vivo and in vitro uses, have a reduced reaction time and be suitable for a genome-wide mapping, even for genome as complex as the human genome.